The broad objectives are to determine the life cycle of Lambornella clarki, (Tetrahymenidae: Ciliophora), to study its natural distribution and infection rates , and to assess its potential as a manipulated biological control agent of the western treehole mosquito, Aedes sierrensis. We also plan to establish L. clarki experimentally in known L. clarki-negative treeholes. Specific lab objectives include development of an in vivo culture system for L. clarki, determining how L. clarki survives in dried-out treeholes between winter rainy seasons, its pathogenesis for larval and adult Ae. sierrenis, and its infectivity and pathogenesis for other mosquito species. The long-term objective include development of an in vitro culture system for L. clarki, and to ultimately develop techniques of laboratory mass-production, storage, and field inoculation of treeholes. Mosquito immatures will be collected from marked treeholes each month and reared under various environmental conditions in labs at Berkeley. Mowquitoes emerging from L. clarki-positive treeholes will be collected in traps and in cages over lab rearing containers. Pathogenesis of L. clarki infections will be studied by phase, SEM and EM study. Some of the spore-like, dessication resistant cysts found in dead larvae will be prepared for microscopic examination, and other such cysts will be treated in different ways and later flooded with treehole water with and without larval rearing factors, etc., and others held to study longevity, mode of infection, etc. Laboratory mass production and field application of this mosquito pathogen could result in the successful use of a biological control agent with excellent potential for self-persistence in treated treeholes. Treeholes and similar container-type mosquito habitats represent breeding sites for some of the most effective mosquito vectors of important human pathogens (e.g. Ae. aegypti and Yellow Fever and Dengue Fever, Ae. polynesiensis and Filariasis, Ae. albopictus and Dengue, and Ae. sierrensis is related to its role as a vector of the dog health importance of Ae. triseriatus and LaCrosse Virus. In California, the known public health importance of Ae. sierrensis is related to its role as a vector of the dog heartworm (Dirofilaria immitis) and the deer body worm (Setaria yehi), both of which occasionally infect humans. It also is a major biting pest of humans in wooded backyards, etc.